Serial Sectioning for 3D Imaging under Scanning Electron Microscopy

by

Devin K. Luu

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science

Graduate Department of Mechanical and Industrial Engineering University of Toronto

© Copyright by Devin K. Luu 2016

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Serial Sectioning for 3D imaging under Scanning Electron Microscopy

Devin K. Luu

Master of Applied Science

Graduate Department of Mechanical and Industrial Engineering

University of Toronto

2016

Abstract

Serial block face scanning electron microscopy (SBFSEM) is a powerful method that

can create 3D reconstructions of large volumes for morphology studies in micro or nano

biological or materials science compared to conventional ultramicrotomy. It allows

visualization of long, dense networked structures with nanoscopic resolutions. Despite

its utility, the adoption rate is low because installing state-of-the-art systems require

extensive modifications to the SEM chamber due to their large size.

This thesis presents a prototype for a plug-and-play SBFSEM system using specially

designed compact micro and nanopositioners that can be placed inside the SEM with no

modifications to the SEM. Through iterations of empirical calculations, computer

simulations, and prototyping, an ultramicrotome capable of cutting thin, uniform

sections was designed. The function of the system was validated by sectioning and

reconstructing a copper TEM grid of known dimensions. The reconstruction was accurate

to within 1% of original dimensions.

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Acknowledgments

Firstly, I would like to thank Professor Yu Sun for inviting me to work in his lab and

introducing me to a new field different from any of my previous work. My experience

with his lab group has influenced my career path for the years to come. Along the same

line, I would like to thank my aunt, Caroline Cao, for stimulating my interest in

engineering and research since childhood.

My project on in-SEM ultramicrotome design was challenging, but with the help of

Douglas Holmyard and Ren Li, who showed me some of the basics of ultramicrotomy, I

learned a lot that would have otherwise taken me much longer to learn on my own.

I would also like to thank all lab members for their help during these two years. Jun

Chen’s knowledge of electronics and his circuit designs gave me a lot of flexibility for

running my system. I would also like to mention Wesley Johnson, Zhensong Xu, Tae Won

Ha and Jun Liu, with whom I formed close relationships.

I received a lot of help from undergraduate summer students. Special thanks to

Tiffany Shan for her assistance in the meticulous set-up of my experiments and to Benn

Xu for performing the time-consuming procedures.

Most importantly, I would like to thank Brandon Chen for always being behind me.

His constant motivation gave me the courage to push ahead in my project during my

many moments of uncertainty. Thank you for your confidence in me and in my work. I

look forward to working with you in the future.

Finally, I would like to thank some people from home in Vancouver for being there

almost around the clock just to chat and take my mind off of work. Also thanks to my

parents and brother not only for their love, but their reminders to take care of my health.

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Table of Contents

Contents

Acknowledgments ...................................................................................................................... iii

Table of Contents ........................................................................................................................ iv

List of Tables ............................................................................................................................... vii

List of Figures ............................................................................................................................ viii

Chapter 1 Introduction .................................................................................................................1

Introduction ..............................................................................................................................1

1.1 Background ........................................................................................................................1

1.2 Motivation ..........................................................................................................................3

1.3 Dissertation Outline ..........................................................................................................4

Chapter 2 Ultramicrotomy in SEM ............................................................................................5

Ultramicrotomy in SEM ..........................................................................................................5

2.1 The SEM Working Environment ....................................................................................5

2.2 Ultramicrotome Function ................................................................................................8

2.3 Cutting in Ultramicrotomy ..............................................................................................9

2.3.1 Tool material and geometry ..............................................................................10

2.3.2 Cutting parameters .............................................................................................11

2.3.3 Sample material ...................................................................................................12

2.4 Existing SBFSEM Systems ..............................................................................................12

2.5 Design of a Plug-and-Play SBFSEM System ...............................................................13

2.6 The Latest Generation Design .......................................................................................15

v

2.6.1 Mechanical description ......................................................................................16

2.6.2 Electrical and software description ..................................................................17

2.7 Conclusion .......................................................................................................................19

Chapter 3 Flexure Based Piezoelectric Actuator ....................................................................20

Flexure Based Piezoelectric Actuator ..................................................................................20

3.1 Introduction .....................................................................................................................20

3.2 Design Considerations for a Flexure Based Piezoelectric Actuator ........................23

3.3 Solving the Out-of-plane Stiffness Problem ................................................................26

3.4 The Designed Piezoelectric Flexure Actuator .............................................................26

3.5 Discussion of Alternative Designs ................................................................................29

Chapter 4 Thermal V-Beam Actuator ......................................................................................30

Thermal V-Beam Actuator ....................................................................................................30

4.1 Introduction .....................................................................................................................30

4.2 Design Considerations of a Thermal V-Beam Actuator ............................................32

4.3 The Designed Thermal V-Beam Actuator ...................................................................34

4.4 Discussion of Previous Specimen Actuator Designs .................................................36

Chapter 5 Sectioning using the SBFSEM System ...................................................................37

Sectioning using SBFSEM System........................................................................................37

5.1 Specimen Height Control for Sectioning .....................................................................37

5.2 Minimum Slice Thickness ..............................................................................................38

5.3 Cutting Surface Roughness ...........................................................................................38

5.4 Cutting Parameters Affecting Surface Roughness .....................................................40

vi

5.4.1 Knife speed has little effect on chatter .............................................................41

5.4.2 Chatter increases as slice thickness increases .................................................41

5.5 Cutting Force Estimation for Future Design Guideline ............................................42

5.6 Oscillating Knife: A Method to Reduce Cutting Force ..............................................43

5.7 3D Reconstruction Accuracy Validation .....................................................................46

Chapter 6 Conclusion and Outlook .........................................................................................50

Conclusion and Outlook .......................................................................................................50

6.1 Contributions ...................................................................................................................51

6.2 Future Directions ............................................................................................................51

References ...................................................................................................................................53

vii

List of Tables

Table 1.1: Some discoveries made using SBFSEM and the volumes reconstructed ........... 3

Table 2.1: Comparison of different actuators. Criteria are listed left to right from most to

least important. ........................................................................................................................... 14

Table 2.2: Specification requirements for a new SBFSEM system ...................................... 15

Table 3.1: Monitored inputs and outputs from simulation are used to check against

specifications ............................................................................................................................... 25

Table 5.1: 3D reconstruction is accurate within 1% for length measurements. ................ 48

viii

List of Figures

Figure 1.1: SBFSEM not only allows automated ultramicrotomy, but it also eliminates

section retrieval, which is considered to be the most difficult step. ..................................... 2

Figure 2.1: In a scanning electron microscope, an electron gun fires electrons that are

focused and directed by a series of lenses and deflectors to perform a raster scan across a

sample. Secondary electrons released from the sample are detected by the detector to

form an image on a video display. ............................................................................................ 6

Figure 2.2: SBFSEM process. A) The knife and specimen are in position and ready to cut.

B) The specimen is cut and the slice is discarded. C) The block face surface is scanned by

SEM. D) The specimen is moved into position for the next cut. ........................................... 9

Figure 2.3: Besides the knife edge, the three angles, rake angle, bevel angle, and clearance

angle play a large role in determining cutting force, and by extension, cutting surface

roughness (chatter) and compression. In general, larger a larger rake angle will give lower

force, but bevel angle and clearance angle need to be balanced to maintain knife longevity

and to prevent rubbing of the knife against the surface, respectively. .............................. 11

Figure 2.4: The ultramicrotome's actuators are mounted on a base plate. A knife is

connected to the knife actuator and the specimen is mounted on the specimen actuator.

....................................................................................................................................................... 17

Figure 2.5: The electronics for the SBFSEM ultramicrotome. The specimen thermal

actuator is run by the bench top power supply on the left and the knife piezo actuator is

run by the amplifier on the right. The amplifier is controlled via serial interface with a

PC. ................................................................................................................................................ 18

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Figure 2.6: A GUI on a PC gives commands to a piezo amplifier for the knife actuator

using serial commands. The interface allows knife speed and knife extension amount to

be set, as well as monitoring of messages. ............................................................................. 19

Figure 3.1: Each solid line represents the set of possible force-displacement operation

points for a given voltage. To maximize the amount of work done by a piezo stack, the

load on the stack should be such that the operation points will lie upon the dashed

maximum work line. ................................................................................................................. 21

Figure 3.2: (Left) Parallel four-bar linkage type flexure actuator. Amplification ratio

depends mostly on the length L, where larger L gives larger amplification ratio. (Right)

Sagittal type flexure actuator. Amplification ratio depends mostly on angle α, where

smaller α gives larger amplification ratio. With the same size envelope, it is easier to

obtain a higher displacement ratio on a sagittal type flexure than a four-bar linkage

flexure. ......................................................................................................................................... 22

Figure 3.3: An example output of an FEA simulation of a piezoelectric flexure mechanism.

The four quantities indicated in the figure should be used as shown in Table 3.1 to

compare with specifications. .................................................................................................... 25

Figure 3.4: (Top) The flexure actuator has a final motion range of 740 µm. (Bottom) There

is a hysteresis effect that is evident on the retraction stroke of the actuator but it is ignored

because no cutting is done on retraction. The extension stroke matches well with

simulation results (less than 5% error).................................................................................... 28

Figure 4.1: In the thermal v-beam actuator, heat is applied, causing thermal expansion of

the flexures. Due to the geometry, the actuator tip will be pushed upwards, as shown

x

with exaggerated displacement. Given a maximum size for the actuator, the displacement

of the tip depends on the thickness and angle θ of the beam. ............................................ 31

Figure 4.2: An example output of an FEA simulation of a thermal v-beam actuator. The

four quantities indicated in the figure should be used to compare with specifications. . 33

Figure 4.3: (Left) For a steady input voltage, after the actuator has reached steady state,

the actuator follows a quadratic voltage-displacement characteristic. The actuator has a

range of 310 µm. (Right) The specimen actuator follows a first-order transient

characteristic with a time constant of 5.23 min and a settling time of 20.5 min inside the

SEM. Knowing this, for any step input voltage to the actuator, it is possible to calculate

the displacement of the actuator at any time. ........................................................................ 35

Figure 4.4: The designed v-beam thermal actuator has a polished, reamed, hole for

accepting a resistive cartridge heater. Flexure beams expand against the bracing, forcing

the actuator tip upwards. .......................................................................................................... 36

Figure 5.1: Timings for five cuts in 20.5 min, with a 500 nm step input. ........................... 37

Figure 5.2: (Top left and right) Optical micrographs of a rough (A) and smooth (B) block

face. The cutting direction is from top to bottom. The rough block face was cut using an

older revision of the ultramicrotome which was not as stiff and severe chatter occurred.

(Bottom) The AFM line scans of the sample show that the RMS of the smooth surface is

substantially lower than the RMS of the rough surface. ...................................................... 39

Figure 5.3: (Left) There is little to no effect of knife speed on chatter. Statistical analysis

shows a low correlation value of -0.45 between knife speed and chatter. (Right) As section

thickness is increased, little chatter until a section thickness of 500 nm, when the

roughness RMS rapidly rises. ................................................................................................... 40

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Figure 5.4: (Top left) A piezoelectric stack is attached to a knife with a counter weight to

oscillate the knife. (Top right) Knife apparent angle, caused by knife oscillation combined

with the knife feed, is smaller than the knife bevel angle. (Bottom left) Block face after

slicing off 400 nm at a frequency of 7 kHz. (Bottom right) Vibrating glass knife becomes

damaged easily, causing severe chatter. The left side of the blade is more damaged than

the right side. .............................................................................................................................. 44

Figure 5.5: (Top left) Individual SEM images were taken, then processed and aligned to

form a stack. (Top right) A TEM grid was reconstructed using SBFSEM with a voxel size

of 66 x 66 x 100 nm. The grid spacing as measured from the 3D reconstruction by our

SBFSEM system is matches with manufacturer specifications. Shaded in areas show the

first section. Outlined areas show debris and discontinuities, as labelled. (Bottom)

Reference TEM grid with dimensions. ................................................................................... 47

1

Chapter 1 Introduction

Introduction

1.1 Background

Serial block face scanning electron microscopy (SBFSEM) is a method combining

conventional ultramicrotomy and scanning electron microscope (SEM) imaging [1].

Ultramicrotomy is an important technique used in life [2, 3] and material sciences [4-6] to

study morphology at the nanometer scale by taking successive thin slices from a sample

[7, 8]. These samples can be imaged and stacked using computer imaging algorithms to

reconstruct a 3D computer model of the sample. This technique can be used to resolve

interior structures that cannot be seen otherwise. Conventional ultramicrotomy is

performed manually by skilled operators and involves several complex steps to cut from

a resin-embedded sample thin slices less than 100 nm in thickness [9]. After the slices are

cut, the operator must painstakingly retrieve each individual section from a small

container of water attached to the knife. Finally, each slice is placed on a transmission

electron microscope (TEM) grid and imaged. Each step must be carefully performed to

ensure that slices do not get lost or wrinkled in the process.

Taking advantage of the large available space inside an SEM, Denk and Horstmann

[1] developed the first working SBFSEM system using custom designed components and

precision actuators tailored for use inside SEM. Their system was capable of

automatically obtaining images from large blocks of tissue. Crucially, their system

eliminated the manual slicing and retrieval steps, which most heavily depend on operator

skill (Figure 1.1). By performing the process under SEM, instead of collecting slices to

image, the slices are discarded and the remaining block face of the specimen is imaged.

The system could automatically section and image specimens unattended, acquiring vast

amounts of data.

2

Owing to these capabilities, many breakthroughs in biological sciences are being

made. Most notably, the morphology of relatively large volumes of tissue or those

containing long, fibrous networks with nanometer-sized features such as neural tissue

[10-18], or muscles [19-21] can now be visualized in detail, giving clues to their functions

and mechanisms. This technology also shows usefulness in other areas of cell biology [22-

28] and materials science [29-36]. Table 1.1 presents a few of these cases and the volumes

reconstructed.

Despite SBFSEM’s capabilities and accomplishments thus far however, the adoption

rate of this new technology is hindered by some factors: Current commercially available

systems are only compatible with certain models of SEM, or require heavy modification

to other SEMs [37, 38]. The cost of a new SEM or extensive modifications is prohibitive to

most microscopy and biology labs. After modification, it may be difficult or even

Figure 1.1: SBFSEM not only allows automated ultramicrotomy, but it also

eliminates section retrieval, which is considered to be the most difficult step.

3

impossible to use the SEM for any other imaging tasks. The cause of this is mostly because

existing systems are large and fill the entire SEM chamber.

The development of other SEM nanorobotic manipulation platforms in more recent

years, such as in sensing, actuation, and automation [39-43], also provides some

technology that could be used in developing a compact SBFSEM system.

Table 1.1: Some discoveries made using SBFSEM and the volumes reconstructed

Category Summary of discovery/application Volume

Reconstructed

Reference

Neuroscience Postsynaptic membrane of most used

synaptic connections are found to be

reinforced with use

80 x 80 x 59.9 µm3 [10]

Synaptic pruning does not proceed

normally in absence of bone

morphogenetic proteins

180000 µm3 [11]

Cell biology A previously unknown ridge-like structure

on podocytes was discovered

2000000 µm3 [22]

Organization of collagen extracellular

matrix is analyzed by SBFSEM

1000 µm depth [19]

Materials

Science

Micro and nanostructures of engineered

materials such as paper and other

composites are reconstructed

60 x 60 x 20 µm3 [29]

1.2 Motivation

Although SBFSEM ultramicrotomes are available commercially, they are large, and

are only available for specific models of SEM. In addition, numerous modifications must

be made to the SEM itself to accommodate the ultramicrotome [37]. Because most electron

microscopy facilities are shared between multiple users, to make modifications to a

microscope would cause great inconvenience for the users. Our goal is to develop a

prototype plug and play SBFSEM ultramicrotome that can be used in any SEM to perform

3D reconstruction of samples.

4

This ultramicrotome will be substantially smaller than other ultramicrotomes on the

market [37]. To accomplish this, existing designs should be studied to understand why

they are so large, and then redesign to work around those limitations. The small actuators

used in other SEM nanomanipulation tasks should be studied and adapted to the new

design. Literature will be reviewed to set specifications for a new ultramicrotome, such

as size, minimum volume able to reconstruct, and other motion requirements. Specific

objectives include:

Study current systems and establish new design specifications.

Design and characterize a new prototype SBFSEM ultramicrotome.

Verify the operation of the designed SBFSEM ultramicrotome by sectioning and

reconstructing structures of known morphology.

1.3 Dissertation Outline

An overview of the following chapters is as follows: Chapter 2 describes the process

of ultramicrotomy, and the integration of that process into SEM. It will also describe the

design challenges and of a compact ultramicrotome that can be used in any SEM. A large

part of the design process involved the development of a piezoelectric flexure and

thermal buckling beam actuator whose designs and characterized performances are

discussed in Chapter 3 and 4, respectively. Chapter 5 presents a demonstration of the

capabilities of the designed ultramicrotome to verify its function. Chapter 6 concludes

the thesis, giving a summary of the thesis, a list of key contribution of this research, and

a list of suggested future directions.

5

Chapter 2 Ultramicrotomy in SEM

Ultramicrotomy in SEM

To design an ultramicrotome for SEM the SEM working environment and

ultramicrotome form and function should be well understood. This section will give an

introduction to the SEM function and ultramicrotome function, then lay out some

guidelines and specifications that will assist in designing ultramicrotomes in SEM, and

other types of designs for SEM in general. Then, it will introduce the designed

ultramicrotome in a general sense.

2.1 The SEM Working Environment

The inside of a scanning electron microscope chamber is kept at high vacuum and

ideally free from outside electric and magnetic fields. Deviations from ideal imaging

conditions can cause artifacts, noise, image warping, or unwanted reflections. Stray

electric and magnetic fields act as lenses to bend the path of electrons. Air remaining in

the chamber will impede the path of electrons, making the signal-to- noise ratio weak.

Hydrocarbons either from outgassing or other contamination like fingerprints will be

deposited onto the specimen by electron beam induced deposition (EBID) [44], obscuring

the specimen. Any defects in the image will interfere with the fidelity of the final 3D

reconstruction [45].

Figure 2.1 depicts the SEM imaging process. When imaging inside SEM, an electron

beam focused by electromagnetic lenses is raster scanned across a specimen surface.

When the electron beam hits the sample, the electrons’ interactions with the sample

produces signals that can be detected by sensors inside the microscope chamber to reveal

information about the sample such as topography or material composition depending on

imaging mode [46].

6

Secondary electron imaging is used to visualize the topography of the sample. When

an incident electron beam hits a spot on the sample surface, secondary electrons are

emitted from the sample, which are then detected by the secondary electron detector.

Backscatter electron imaging is used to visualize composition information of the sample.

When an incident electron beam hits a spot on the sample surface, electrons are slung

back from the specimen. The number of electrons slung back depends on the atomic

number of the area of the specimen, with higher atomic numbers slinging back more

electrons [46].

Figure 2.1: In a scanning electron microscope, an electron gun fires electrons that are focused and

directed by a series of lenses and deflectors to perform a raster scan across a sample. Secondary

electrons released from the sample are detected by the detector to form an image on a video display.

7

Knowing the basic operation of the SEM and about factors that affect its operation,

here are a few material selection and handling factors that all designs for SEM should

consider:

1. Outgassing – Certain plastics will have gas pockets trapped within during

manufacturing. The released gases may interfere with electron paths, and in the

worst case, can be deposited on specimen surfaces via EBID [45]. Aluminum alloys

and 300 series stainless steels are common choices for metals, while PTFE, PEEK,

and Kapton are common choices for plastics used in SEM. Special lubricants and

adhesives are also manufactured to be low outgassing. Similarly, before entering

the SEM chamber, all materials should be thoroughly cleaned to remove

contamination such as fingerprints. A comprehensive list of material outgassing

properties can be found at [47].

2. Magnetic fields – Magnetic materials may magnetize and bend the path of

electrons, causing distortion in the image. Metals used that exhibit little or no

magnetism are 300 series stainless steels and aluminum alloys. In the case that

magnetic materials must be used, effort should be spent on placing these materials

as far away as possible from the imaging region of interest.

3. Specimen charging – Non-conductive materials will “hold on” to electrons as the

electron beam is scanned over them, causing distortions/bright spots in image.

Conductive materials provide a path for excess electrons to leave the sample to

ground. Ideally, any specimens under SEM should have a conductive path to

ground, and any non-conductive materials, like with magnetic materials, should

be placed as far away from the imaging area of interest as possible.

Also important is that although the SEM chamber is large, the instruments and

detectors within are extremely delicate and sensitive. Any specimens or machines that

8

are put inside the SEM must fit well, such that they are secure and do not have a risk of

coming loose and falling and hitting any SEM emitters or detectors.

In SBFSEM, to accommodate the non-conductive resin-embedded sample, a special

variable pressure or low vacuum mode, which introduces small amounts of gas into the

chamber is used to whisk away excess charge. BSE imaging is used to obtain large image

contrast between different materials in the block face surface [1].

2.2 Ultramicrotome Function

A conventional ultramicrotome is a large desktop machine, incorporating a

microscope a cantilever arm, a specimen holder, a glass or diamond knife, and a knife

holder [48]. In conventional ultramicrotomy, the specimen is mounted on a cantilever

arm which moves the specimen face across a stationary knife edge when a crank wheel

is turned, which is held on the knife holder stage [49]. To take successive cuts, a heating

element on the cantilever arm causes the arm to thermally expand, advancing the

specimen towards the knife by a small amount [49]. Each part must be mounted as rigidly

as possible to withstand cutting forces to ensure uniform, thin slices of sample [50]. After

cutting, the slice floats away on the surface of water in a container attached to the knife

for retrieval and imaging in TEM.

The process of SBFSEM is shown in Figure 2.2 and is also introduced in [1]. Besides

being automated, the key mechanical difference between the conventional

ultramicrotome and SBFSEM is that instead of the specimen both advancing and

providing a slicing motion, it only advances toward the knife. The knife now provides

the cutting motion itself. The reason for this is to keep the specimen block face still with

respect to the SEM image frame. By advancing the specimen each time instead of moving

the knife to a different level, the focal plane of the specimen block face will also stay the

9

same. This allows the sliced section to be discarded, while imaging the remaining block

face.

2.3 Cutting in Ultramicrotomy

The sectioning process in ultramicrotomy is a cutting process comparable to

orthogonal cutting in traditional machining. There are a few factors in ultramicrotomy

that will affect the quality of the cut, including the material and geometry of the tool, the

cutting parameters, and the material [9, 50]. In ultramicrotomy, some concerns are chatter

and compression [50]. Chatter manifests as marks running perpendicular to the cutting

directions along the block face, and causes sections to come off which are uneven in

thickness. Compression occurs when the section coming off the block face is thicker than

the original depth of cut. A cut which requires low force to make results in lower

compression [51, 52], and a rigid setup results in lower chatter [50, 53]. In SBFSEM, where

we are only observing the block face, compression is not a big concern, but chatter will

still decrease the accuracy of the reconstructed volume.

Figure 2.2: SBFSEM process. A) The knife and specimen are in position and ready to cut. B) The

specimen is cut and the slice is discarded. C) The block face surface is scanned by SEM. D) The specimen

is moved into position for the next cut.

10

2.3.1 Tool material and geometry

The tool is affected by its material and geometry. In ultramicrotomy, metal knives

such as razor blades are not used because they become blunt too quickly. Instead, glass

and diamond knives, which also have very small edge radii are used. Compared to

diamond knives, glass knives have a slightly larger edge radius, their edge is not as

straight, and they are not as durable. Furthermore, glass knife edges’ sharpness and

straightness will degrade over time [9].

The geometry of the knife is also important to the cutting quality [54]. The rake angle,

the bevel angle, and the clearance angle are three important parameters to consider about

ultramicrotome knives [55]. The three of these angles add up to a right angle, as shown

in Figure 2.3. Because they play a large role in cutting force, their role should be

considered in the overall ultramicrotome design to mitigate chatter, which arises when

the structure is not stiff enough to withstand the cutting forces. High forces also

contribute to compression, which is that the slice after cutting is thicker than the slice

before cutting.

When looking at a 2D cross section of the knife and specimen, the rake angle is the

angle made by the top of the knife and the line normal to the specimen. A smaller rake

angle gives smaller cutting force. The bevel angle is the angle of the knife itself. Although

a smaller bevel angle can help increase the rake angle, a bevel angle that is too small will

cause the knife to be very fragile. The clearance angle is the angle made by the bottom of

the knife and the specimen, and its purpose is to prevent rubbing of the bottom of the

knife with the specimen. Diamond knives typically have a smaller bevel angle, allowing

the rake angle to be larger [52, 56]. A glass knife, having a larger bevel angle is more

forgiving against mistakes during setup.

11

2.3.2 Cutting parameters

Cutting parameters that affect the cut quality are the width of cut, depth of cut, and

speed of cut. The cutting force is proportional to the width of the cut [55]. The depth of

cut also affects the cutting force [55]. The thicker the depth, the more force it takes to curl

the cut section over the edge of the knife as it moves over the block face [55]. In extreme

cases, this can cause parts of the sample to tear off or it will break the knife [9]. Cutting

speed that is too high can result in excess chatter because any vibrations resulting from

the knife engaging the sample do not have enough time to damp out before the knive

begins to move across the block face [9]. Cutting too slow can also potentially cause the

Figure 2.3: Besides the knife edge, the three angles, rake angle, bevel angle, and

clearance angle play a large role in determining cutting force, and by extension, cutting

surface roughness (chatter) and compression. In general, larger a larger rake angle will

give lower force, but bevel angle and clearance angle need to be balanced to maintain

knife longevity and to prevent rubbing of the knife against the surface, respectively.

12

resin to stick to the edge of the knife [57], temporarily making the knife edge effectively

less sharp.

2.3.3 Sample material

Different embedding media, as well as the specimen embedded inside the embedding

media, exhibit different sectioning properties. For example, the hardness, stiffness, and

the material’s microstructure can affect cutting quality. Also very important is the

specimen preparation, including whether or not the embedding medium has been

properly cured and whether it has fully penetrated the specimen embedded inside [58].

2.4 Existing SBFSEM Systems

There are two main commercial systems for performing SBFSEM on the market: Gatan

3View2 [37] and FEI Teneo VS [38]. Although both products have been demonstrated to

be useful, they are currently compatible only with select models of SEM, or only after

heavy modification to the SEM.

The Gatan 3View2 system [37] is based on the work in [1]. The mechanism is based

on a series of cams and motors, requiring the use of multiple motion transfer components,

making the system large. Therefore, to heavy modifications are required to integrate the

3View2 into the SEM. The microtome is mounted permanently directly onto the SEM

door.

The FEI Teneo VS [38] is a different system utilizing a technique called multi-energy

deconvolution [59] to be able to potentially resolve thinner slices than a knife can

physically cut by using the electron beam to penetrate deeper than the surface of the

sample, then subtracting the surface information from the signal. However, any

variations in the material in the block face can potentially make the image slices uneven.

This ultramicrotome is only available to be fitted in one specially made SEM because it

also uses large mechanical components.

13

In conclusion, the main factor limiting the commercial SBFSEM systems is that they

are too large because they use a series of large mechanical components to transfer motion.

After SEM modification and installation, it may be difficult or even impossible to use the

SEM for other imaging tasks.

2.5 Design of a Plug-and-Play SBFSEM System

A SBFSEM ultramicrotome needs to be able to hold a knife and move the knife edge

over a cutting plane. It also needs to be able to hold a specimen tightly and to move the

specimen surface into the knife cutting plane. The described motions need to be high

resolution, smooth, and repeatable. The structure must be able to withstand cutting

forces, meaning the structure must be stiff. All the components used in its construction

must also be vacuum compatible.

There are a few additional challenges in designing a compact SBFSEM system. The

greatest challenge in developing a miniaturized system is that a smaller system will have

lower stiffness. In a system like an ultramicrotome, where not only nanometer

positioning is required, but the positioners also need to withstand cutting forces, even a

deflection of a several nanometers is crucial. Insufficient stiffness will result in

inconsistent slice thicknesses and also chatter knife marks.

Although conventional mechanical components such as motors, bearings, and gears

can achieve high stiffness and high speed, they will not be used to generate the

ultramicrotome motions because they are too large and there is risk of mechanical play.

This requirement rules out other piezoelectric actuators such as inchworm, walking, or

stick-slip mechanisms, which essentially use piezoelectric elements to generate pushing

motions along a bearing rail. The requirement against magnetic fields will rule out

magnetic actuators. Instead, flexure based actuators driven by piezoelectric stacks or

thermal expansion, which by nature have a certain amount of compliance to generate

14

required motions, will be used, adding to the stiffness challenge. Great care is needed to

balance the stiffness and motion requirements. A summary of different actuator types is

shown in Table 2.1.

Table 2.1: Comparison of different actuators. Criteria are listed left to right from most to least important.

Actuator

Type

Requirements Other metrics

Non-magnetic Repeatable Motion

range

Stiffness Score

Motors/gears FAIL FAIL

Magnetic FAIL Pass

Piezo walk /

slip stick

Pass FAIL

MEMS Pass Pass -- - -3

Piezo stack Pass Pass -- + -1

Piezo

flexure

Pass Pass DATUM 0

Thermal

flexure

Pass Pass - + 0

The ultramicrotome will use a glass knife due to availability and cost. Although glass

knives are inferior to diamond knives in almost every aspect in sectioning, the cost of a

glass knife is much lower, making it suitable for prototyping. The higher bevel angle

(~50°) of a glass knife also makes it less fragile, making it more forgiving to mistakes

during setup. For now, the SEM to be used with this design is the Hitachi SU3500, which

was picked because of availability.

To quantify the above functional requirements, the end performance target

specifications were set as follows in Table 2.2. The maximum system size was set to be

the maximum size a system can be and still be safely integrated into the SEM. Based on

the sizes of the reconstructions shown in Table 1.1, the maximum sample block face size

and advance distance were set to be able to cover the volumes reconstructed in

neuroscience and materials science. The knife actuator range was set to allow the entire

15

block face to be cut. The minimum slice thickness for this system is set near the minimum

thickness typically cut with glass knives on a conventional ultramicrotome [9].

Table 2.2: Specification requirements for a new SBFSEM system

Aspect Our target specification

System size < 127 x 127 x 90 mm3

Block face size < 350 x 350 µm2

Sample advance

distance

> 250 µm

Knife travel distance > 700 µm

Slice thickness for glass

knife

< 100 nm

The ultramicrotome underwent several iterations before arriving at the latest design.

The first versions used exclusively piezoelectric actuators due to ease of control, and were

focused on generating the correct motions. However, the initial designs of the

piezoelectric actuators were not stiff enough, causing sever chatter while cutting. The

latest generation focused on maximizing the stiffness of the system to achieve less chatter

while sectioning.

2.6 The Latest Generation Design

In the latest design iteration, keeping in mind the requirements for the system, and

especially trying to preserve the stiffness of the small system, the following system was

designed. The most drastic change from previous iterations was that the specimen

actuator was changed from a piezoelectric actuator to a thermal actuator, sacrificing ease

of control and speed for stiffness. The aspects that most affected the design of this system

was the actuator design, and they will be discussed in further detail in later chapters.

16

2.6.1 Mechanical description

Most structural mechanical parts are custom machined from 6061 aluminum alloy or

304 stainless steel, which are non-magnetic. All actuators are mounted on a common

baseplate. Any parts that may need to slide along each other are manufactured out of

different materials to eliminate any possibility of cold welding inside the SEM vacuum

chamber.

As shown in Figure 2.4, a piezoelectric knife actuator, which performs a slicing motion

by pushing the knife horizontally, is attached to the glass knife holder which holds a knife

with a clearance angle of 6° to prevent rubbing of the knife bottom while cutting [9]. A

thermal specimen actuator, which moves the specimen vertically into position for cutting

before each slice, is attached to the specimen holder. The specimen holder is separated

from the thermal actuator by a piece of Mycalex MM400, which insulates the specimen

from the heat generated by the specimen actuator. All electrical connections for the

actuators are routed to one connector which will be fed outside the SEM chamber via a

specially designed SEM feedthrough port flange. All wires and piezoelectric actuators are

shielded to avoid noise in the SEM image. Wire jackets are made of PTFE to avoid

outgassing.

17

2.6.2 Electrical and software description

The piezoelectric knife actuator is powered by an amplifier custom-designed to run

piezoelectric actuators. The amplifier was designed by Jun Chen (PhD candidate). The

thermal actuator is controlled simply by a B&K Precision 9110 bench-top power supply.

The two power sources are shown in Figure 2.5. Both actuators are controlled in open

loop after careful characterization within the SEM chamber.

Figure 2.4: The ultramicrotome's actuators are mounted on a base plate. A knife is connected to

the knife actuator and the specimen is mounted on the specimen actuator.

18

The microtome can be controlled from a GUI that interfaces with the amplifier

through USB, shown in Figure 2.6. The interface provides a way to control the speed and

travel of the knife as well as a way to monitor the status of the ultramicrotome while it is

moving.

Figure 2.5: The electronics for the SBFSEM ultramicrotome. The specimen thermal actuator is run by

the bench top power supply on the left and the knife piezo actuator is run by the amplifier on the right.

The amplifier is controlled via serial interface with a PC.

19

2.7 Conclusion

In summary, a system was designed and manufactured to target the specifications

laid out for the compact ultramicrotome system. The specifications of the designed

system were able to slightly exceed their targets.

The following sections will present a more in-depth discussion of the design and

characteristics of the main components and actuators of the system.

Figure 2.6: A GUI on a PC gives commands to a piezo amplifier for the knife actuator using

serial commands. The interface allows knife speed and knife extension amount to be set, as well

as monitoring of messages.

20

Chapter 3 Flexure Based Piezoelectric Actuator

Flexure Based Piezoelectric Actuator

A flexure based piezoelectric actuator was used to actuate the knife. The actuator

technology used must exhibit smooth, vibration free, and repeatable motion. The motion

range should be at least 700 µm to fully cover the previously determined block face size

of 350 µm x 350 µm. The out-of-plane stiffness should be at least 2.2 N/µm to ensure less

than 20 nm of displacement for a vertical forces of 50 mN induced during cutting [60]. It

should also be able to output approximately 50 mN of force through the whole range that

it is cutting. The actuator was designed to be as compact as possible.

This chapter discusses the background and design considerations for a piezo actuator,

then it presents a design for the piezoelectric flexure actuator that was used in the

SBFSEM ultramicrotome. Finally, alternative solutions are discussed, focusing on why

they were not used in this design.

3.1 Introduction

Piezoelectric actuators operate based on the inverse piezoelectric effect [40], meaning

an electric field applied to them will cause a deformation. They are a popular choice for

actuators inside SEM because they are easy to control in that they exhibit a near linear

voltage-displacement characteristic, and are capable of high force and nanometer

resolution. Many piezoelectric actuators are available in the form of stacks [61, 62], where

layers of piezoelectric material are layered to combine their displacements to create a

larger motion range.

Piezoelectric stack actuators can in one way be characterized by the following lines in

Figure 3.1 [63]. Each line represents a set of possible force-displacement outputs for a

21

given input voltage, the operation point of the actuator can be found given the load. There

are two quantities of interest:

Free stroke of the actuator is the x-intercept of the line and corresponds to the

displacement when the stack is actuated without load.

Blocked force is the y-intercept of the line and corresponds to the actuator’s force

output when the actuator is compressed back to zero displacement from the free

stroke length.

One implication is that any operation point beyond the line for the maximum voltage

of the piezoelectric stack cannot be reached. Another implication is that to maximize the

amount of work done by the piezo, the operation points of a piezoelectric driven device

should lie upon the maximum work line. This line represents the most balanced force-

Figure 3.1: Each solid line represents the set of possible force-displacement operation points

for a given voltage. To maximize the amount of work done by a piezo stack, the load on the

stack should be such that the operation points will lie upon the dashed maximum work line.

150 V

120 V

90 V

60 V

30 V

Maximum work line

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 10 20 30 40 50

Availa

ble

Outp

ut

Forc

e [N

]

Displacement [µm]

22

displacement operation. This information is important when designing actuators to

ensure that force and displacement requirements are satisfied.

To further amplify their motion, piezoelectric stack actuators are often placed within

a compliant flexure mechanism, consisting of thin flexures acting as hinges and thicker

rigid links between flexures [64]. There is no sliding motion between any surfaces in a

flexure mechanism, so a piezoelectric actuator can achieve perfectly smooth, repeatable

motion. There are chiefly two different types of flexure mechanisms for a flexure: the

parallel four-bar linkage type, and the sagittal type shown in Figure 3.2 left and right,

respectively. In both cases, a small horizontal expansion of the piezoelectric element will

cause bending of the thin flexures, leading to an amplified vertical displacement of the

actuator tip. The amplification depends on length L for the parallel bar flexure, and angle

α for the sagittal flexure.

Although the motion is greatly amplified, the final force output and stiffness of the

actuator is also greatly reduced compared to the piezo stack itself. Furthermore, the

Figure 3.2: (Left) Parallel four-bar linkage type flexure actuator. Amplification ratio depends

mostly on the length L, where larger L gives larger amplification ratio. (Right) Sagittal type

flexure actuator. Amplification ratio depends mostly on angle α, where smaller α gives larger

amplification ratio. With the same size envelope, it is easier to obtain a higher displacement ratio

on a sagittal type flexure than a four-bar linkage flexure.

23

higher the amplification ratio the higher force is required from the piezoelectric stack

because the smaller α increases the flexure’s stiffness from the stack’s perspective.

Therefore, although it is possible to have very high amplification ratios, the application

of the actuator should be analyzed carefully to determine the force requirements.

The two types of actuators differ in that it is easier to achieve a larger amplification

ratio on a sagittal type flexure, according to computer simulations. Also, the sagittal

actuator tip will move more in a straight line while the four bar linkage actuator tip will

move more in an arc. However, reasons for using a parallel four- bar linkage type flexure

include that they have different mounting points, which may be more convenient based

on the application.

3.2 Design Considerations for a Flexure Based Piezoelectric Actuator

The sagittal type actuator was chosen for this design because of its larger amplification

ratio for the same size envelope and its mounting point was deemed more convenient.

Furthermore, to design an actuator of the same amplification ratio in the four-bar linkage

style would make the actuator too large. An effect of that would be that the actuator

would act as a large overhanging cantilever, decreasing the stiffness.

The reason that the sagittal type design allows for larger amplification ratio compared

to a parallel four-bar linkage flexure of the same size is because it depends the angle of

the “hinges” in the flexure. Generally, the smaller the angle, the larger the amplification

ratio. Conversely, the amplification ratio of a four-bar linkage type flexure depends on

the length of the bars running parallel to the piezo stack, indicated in Figure 3.2.

Knowing the actuator’s target displacement, the piezoelectric stack’s displacement,

and the maximum size of the actuator, an amplification ratio can be selected and a

formula (Equation 3.1) [64] can be used to calculate the required angle between flexures:

24

Where G is gain, Δy and Δx are the flexure’s displacement and piezo stack’s

displacement, respectively, L is length of the bar between the hinges, and α is the angle

between the hinges.

However, a larger amplification ratio will cause the actuator to be stiffer in the

direction the piezoelectric stack is pushing while being softer in the direction of actuation.

Therefore, keeping in mind the operation points shown in Figure 3.1, if the flexure is too

stiff in the stack’s actuation direction, the actuator will exhibit very low displacement,

regardless of the amplification ratio.

In a flexure mechanism, the thin flexures do not exactly emulate hinges, so to obtain

the exact amplification ratio, it is necessary to perform finite element analysis (FEA) to

ensure that the required motion is achieved. In the analysis, it is appropriate to treat the

flexure mechanism as a static structural component. In performing the FEA, the main

input load is the the piezoelectric stack’s exerted force (F) and the outputs are the

piezoelectric stack’s displacement (Δx), the flexure’s displacement (Δy), and the von

Mises stress (σ) of the structure, as shown in a example solution in Figure 3.3. These

quantities are used to check the simulation results against specifications, as shown in

Table 3.1.

Equation 3.1: Assuming flexures are pin joints, this equation can be

used to calculate flexure angles to obtain the needed amplification ratio

𝐺 =∆𝑦

∆𝑥= √

𝐿2

∆𝑥2 sin2 𝛼+

2𝐿

∆𝑥𝑐𝑜𝑠𝛼− 1 −

𝐿

∆𝑥𝑠𝑖𝑛𝛼

25

Table 3.1: Monitored inputs and outputs from simulation are used to check against specifications

To check Use Do

Amplification

ratio

Stack displacement, Δx

Flexure displacement, Δy

Δy/ Δx

Operation

point

Stack displacement, Δx

Stack exerted force, F

Plot on Figure 3.1 to ensure

operation point is within the

stack’s capabilities

Motion range Flexure displacement, Δy Ensure that the specified

motion range is reached

without exceeding maximum

operation point

Stress Von Mises stress, σ Ensure that stress is below

yield stress for selected

flexure material

It is especially important to ensure that the operation point is possible with the

piezoelectric stack to be used in the design. It is also important to ensure that the stress

actuator structure does not exceed its yield stress. To change these outputs, the

Figure 3.3: An example output of an FEA simulation of a piezoelectric flexure mechanism. The four

quantities indicated in the figure should be used as shown in Table 3.1 to compare with specifications.

26

parameters of flexure angle, flexure thickness, and flexure length should be altered. In

general, increasing each parameter will cause amplification ratio and the stack exerted

force to decrease, but will also cause stress to decrease. If no combination of those

parameters is possible to achieve the required amplification ratio and motion range, it

may be necessary to increase the overall size of the flexure structure.

Finally, it is important to consider the mounting of the piezoelectric stack within the

actuator. To ensure that the piezoelectric stack maintains good contact during operation

when placed within the flexure, the stack must be preloaded [63]. Because piezoelectric

stacks are brittle, the loading must be evenly distributed on the ends and parallel to the

stack to avoid any torsion or bending of the stack. Internal tensile forces, which arise from

torsion and bending, will cause premature failure of the stack.

3.3 Solving the Out-of-plane Stiffness Problem

Through simulations, it was found that the flexure actuators were not stiff enough in

the direction perpendicular to the block face. This means it cannot withstand the forces

exerted vertically while cutting. This will cause sever chatter as discussed in section 2.3.

To remedy this, an extra flexure was added to brace the actuator in that direction.

3.4 The Designed Piezoelectric Flexure Actuator

The piezoelectric stack (PiezoDrive SA070742), with a free stroke and blocking force

of 50 µm and 1800 N, respectively, was chosen for this actuator because it has the largest

blocked force and free motion range among commercial piezoelectric stacks. To reach the

specification of 700 µm amplified displacement from 50% of the free stroke to achieve

maximum work, the formula was applied initially to find a hinge angle for an

amplification ratio of 28.

27

The present piezoelectric flexure mechanism was manufactured from 304 stainless

steel, which is non-magnetic. Other popular alternatives for manufacturing flexures are

6061 aluminum and Ti-6Al-4V titanium alloy, but they are not as stiff as 304 stainless

steel. All of these materials are suitable for machining using electric discharge machining

(EDM), which is the typical way of manufacturing piezoelectric flexure mechanisms.

The preloading of the piezoelectric stack is applied using a set screw. To prevent

torsional forces from being transmitted to the piezoelectric stack, the screw bears on a

thin steel plate placed between the stack and the screw. While preloading, the plate is

prevented from spinning with the screw. The plate also acts to evenly distribute the

preload force over the end of the piezoelectric stack.

FEA simulations showed that the piezoelectric stack did not have enough force to

achieve 700 µm with the desired amplification ratio. Therefore, the amplification ratio

was lowered to allow the piezoelectric actuator to expand more. This also allowed the

piezoelectric stack to overcome the stiffness added in the actuating direction by the

support flexure. In adding the support flexure, simulations showed that an increase in

stiffness in the actuating direction corresponds approximately to a 1:1 decrease in motion

range. In this case, an increase in stiffness of 40% lead to a decrease in motion range of

40%. The support flexure increased the out of plane stiffness of the actuator by about 50

times while still allowing the motion range requirement to be met.

In the end, iterations of simulations guided the detailed dimension’ selection in the

design of this actuator. The overall size was made to be as small as possible while

accommodating the piezo stack and satisfying the motion, stiffness, and stress

requirements as discussed in 3.2 and Table 3.1. At the same time, it was ensured that the

thin features’ dimensions could still be reliably manufactured by EDM.

28

The flexure mechanism has a motion range of 740 µm with an amplification ratio of

21.3, as shown in Figure 3.4.

The out-of-plane stiffness is 15.2 N/µm, which is well over the minimum required

amount, and the blocked force is 20 N, which is more than is needed to slice [60]. Without

the support flexure, the out of plane stiffness is 0.333 N/µm. This motion range matches

the simulation result of 780 µm to within 5%, showing that this simulation model can

Figure 3.4: (Top) The flexure actuator has a final motion range of 740 µm. (Bottom) There is a

hysteresis effect that is evident on the retraction stroke of the actuator but it is ignored because no

cutting is done on retraction. The extension stroke matches well with simulation results (less than

5% error).

Extend

Retract

Maximum displacement150 V, 740 µm

Simulation

0

100

200

300

400

500

600

700

800

0 50 100 150

Dis

pla

cem

ent

[µm

]

Input Voltage [V]

29

provide a good guideline for future actuators of similar design. This error can be

attributed to small errors in alignment and dimensions arising from the manufacturing

and assembly processes.

The piezoelectric flexure exhibits a hysteresis curve typical for piezoelectric actuators.

The extension stroke follows a quite linear voltage-displacement characteristic, while the

retraction stroke exhibits a maximum of 20.3% non-linearity error in its voltage-

displacement characteristic. The non-linearity of the retraction stroke does not affect the

operation of the ultramicrotome because the knife itself moves along the same path

during extension and retraction. Therefore, the hysteresis is not compensated for and the

knife is run in open loop. However, to reduce the effects of hysteresis while operating in

open loop, feedforward drives [65, 66] or charge drives [67, 68] can be implemented.

3.5 Discussion of Alternative Designs

In previous designs of the flexure actuator, two piezo stacks were placed in series in

an attempt to increase the motion range while using the “maximum work line”. Although

in theory and simulation, this is possible, it is risky to implement. Unless the stacks are

assembled with high accuracy, it is very easy to misalign and place unintentional tensile

loads on the stacks, causing them to fail. A rule of thumb is that when placing

piezoelectric stacks in series, the end side length should be more than one-tenth of the

total length after stacking.

Another option to increase motion range is to place two flexure actuators in series.

Although this method avoids having two piezo stacks in series, the tip of the actuator is

now a large overhanging beam, meaning the stiffness is very low. Therefore, the

“maximum work line” is not used and only a single actuator with a single stack is used.

30

Chapter 4 Thermal V-Beam Actuator

Thermal V-Beam Actuator

The thermal V-beam actuator is used as the actuator to advance the specimen. In

addition to having smooth and repeatable motion, this actuator also needs to be stiff. The

displacement of the actuator needs to be at least 250 µm to satisfy the specimen advance

distance requirement. The stiffness in the specimen advance direction of the actuator

needs to be 2.2 N/µm, similar to the knife actuator. The maximum temperature that the

actuator should reach is 90 °C, which will be safe for the SEM stage.

This chapter discusses the background and design considerations for a thermal v-

beam actuator, then it presents the design for the v-beam actuator that was used in the

SBFSEM ultramicrotome. Finally, alternative solutions are discussed, including a

comparison between the thermal actuator and a piezoelectric actuator of similar size and

motion specifications.

4.1 Introduction

Thermal V-beam actuators are also flexure based, allowing smooth, repeatable

motion. Instead of an element placed inside the actuator like in the case of piezoelectric

flexure actuators however, the movement is caused by thermal expansion of the

actuator’s flexures, which are angled. The flexures warm up and expand, while the

relatively thicker parts of the actuator remain cool to brace against the expansion, forcing

the actuator tip to move as shown in Figure 4.1. The temperature-displacement

characteristic of the actuator is roughly linear. The thermal aspect of the actuator allows

for very smooth, high resolution motion while the bracing aspect of the actuator allows

high force output.

31

Similarly to the piezoelectric actuator, a small displacement, now caused by thermal

expansion, translates to a large displacement at the actuator tip. A smaller flexure angle

also translates to a larger displacement. Furthermore, a larger displacement also leads to

lower stiffness in the actuating direction. Differently from piezoelectric actuators, it is

possible to place many flexures in parallel to compensate for the lower stiffness. Because

it is the flexures themselves and not an external component that cause the displacement,

meaning force is not as big of a concern. However, more heat will be needed to cause the

same thermal expansion in more flexures.

Conventionally, this type of actuator was used in microelectromechanical systems

(MEMS) [69], and heating is provided by running current directly through the actuator’s

Figure 4.1: In the thermal v-beam actuator, heat is applied, causing thermal expansion of the flexures.

Due to the geometry, the actuator tip will be pushed upwards, as shown with exaggerated

displacement. Given a maximum size for the actuator, the displacement of the tip depends on the

thickness and angle θ of the beam.

32

flexure beams, resulting in resistive heating. Heating power has a quadratic relationship

with input voltage, and is given by Equation 4.1.

However, for this macro scale actuator, the low resistance of the flexure makes it

unfeasible to heat up that way. Instead, a resistive cartridge heater is used. This allows

more freedom in varying the geometry of the actuator because heating is no longer tied

to the resistance of the actuator. Also contrasting with the MEMS version of the actuator

is that because the thermal mass is so much higher, it is more difficult to control due to

its slow speed and sensitivity to ambient temperature variations.

4.2 Design Considerations of a Thermal V-Beam Actuator

In SBFSEM, the maximum temperature of the thermal actuator should be selected so

that it does not damage the SEM stage. Also, care should be taken to isolate the sample

and the knife from the heat generated by the thermal actuator. Knowing the desired

displacement of the actuator tip, the maximum size of the actuator, and the maximum

temperature desired, the angle for the flexure beams can be determined from Equation

4.2 [70]:

Equation 4.1: Resistive heating power is caused by ohmic

heating, which has a quadratic relationship with voltage.

𝑃 =𝑉2

𝑅

Equation 4.2: Assuming uniform flexure beam temperature, this equation can be used

to calculate the angles of the flexure beams required to generate the needed displacement.

𝑈 = 𝛼∆𝑇𝑙𝑠

(𝑠2 + 𝑐2 12𝐼𝐴𝑙2)

33

Where U is the displacement, α is the thermal expansion coefficient of the material, ΔT is

the change in temperature, l is the length of the beam, I is the second moment of area of

the beam, and A is the cross sectional area of the beam. The s and c are shorthand for sinθ

and cosθ where θ is the angle of the flexure beams, as shown in Figure 4.1.

Because the beam temperature is not uniform and there is a temperature gradient

along the beam, it is necessary to run FEA simulations to confirm the geometry. The

thermal actuator should be modeled as a thermal-structural component. The input load

is a thermal flux (Q) with units of power divided by area to where the cartridge heater is

placed, and the outputs are the temperature (ΔT), the tip displacement (U), and the von

Mises stress (σ). Because of the vacuum environment and the relatively low temperatures

reached by the actuator, convection and radiation heat transfer are ignored.

The thermal flux (Q) should not exceed the maximum power output of the cartridge

heater to be used. The change in temperature (ΔT) should not exceed the safe temperature

for the load on the actuator tip, or in the case of the SBFSEM ultramicrotome, cause

Figure 4.2: An example output of an FEA simulation of a thermal v-beam actuator. The four quantities

indicated in the figure should be used to compare with specifications.

34

damage to the SEM stage. The tip displacement (U) should meet the displacement

requirement. Von Mises stress (σ) should be under the yield stress for the given material.

When designing the v-beam actuator, consideration should be given to the mounting

of the heating cartridge. Most resistive heating cartridges are meant to be placed inside a

hole. The hole should be reamed to an accurate diameter and smooth surface finish, with

thermally conductive compound applied to maximize the efficiency of heat transfer

between the heater body and the actuator.

4.3 The Designed Thermal V-Beam Actuator

The designed thermal v-beam actuator uses the ThorLabs HT15W, with a resistance

of 38.4 Ω. With an input voltage of 16.5 V, it has a motion range of 310 µm, exceeding the

250 µm specimen travel distance requirement and staying under the 15 W maximum

output of the heater, as shown in Figure 4.3. The maximum temperature reached to

achieve this motion range is 70 °C, which is safe for the SEM stage. The stiffness of the

actuator in the actuating direction and the cutting direction is 2.21 N/µm and 58.8 N/µm,

respectively. The actuator is shown in Figure 4.4.

The actuator moves slowly given a voltage input and follows a first order transient

response. The time constant is 5.23 min, while the settling time is 20.5 min, as shown in

Figure 4.3. However, knowing the transient characteristic, it is possible to give a large

step input and know the position of the actuator tip at any given time. For example,

instead of inputting a step voltage to displace the actuator 100 nm and waiting for 20.5

min, a step voltage can be given to displace the actuator 500 nm, and the time intervals

for each 100 nm within the 500 nm will be known.

35

The thermal v-beam actuator is manufactured from 304 stainless steel and was

machined using EDM. 6061 aluminum alloy’s combination of heat capacity and thermal

conductivity cause the entire structure to be roughly uniform in temperature, meaning

the thicker parts of the actuator do not stay cool and brace against the expansion of the

flexure beams.

Figure 4.3: (Left) For a steady input voltage, after the actuator has reached steady state, the actuator

follows a quadratic voltage-displacement characteristic. The actuator has a range of 310 µm. (Right)

The specimen actuator follows a first-order transient characteristic with a time constant of 5.23 min

and a settling time of 20.5 min inside the SEM. Knowing this, for any step input voltage to the

actuator, it is possible to calculate the displacement of the actuator at any time.

16.5 V, 310 µm

0

50

100

150

200

250

300

350

0 5 10 15 20

Dis

pla

cem

ent

[µm

]

Input Voltage [V]

Displacement

Fitted

Time constant,

τ = 5.23 min

Settling time,Ts = 20.5 min

0

20

40

60

80

100

0 10 20 30

Dis

pla

cem

ent

[% fin

al valu

e]

Time [Minutes]

Displacement

Fitted

36

4.4 Discussion of Previous Specimen Actuator Designs

In earlier iterations of the ultramicrotome, the specimen actuator used a piezoelectric

flexure design as discussed in chapter 3. According to FEA simulations, the actuating

direction stiffness of the piezoelectric actuator was 0.0429 N/µm, compared to the 2.21

N/µm of the thermal actuator, even though the two actuators have similar size envelopes.

As shown in Figure 5.2, the low actuator stiffness causes severe chatter while sectioning.

Figure 4.4: The designed v-beam thermal actuator has a polished, reamed, hole for accepting a resistive

cartridge heater. Flexure beams expand against the bracing, forcing the actuator tip upwards.

37

Chapter 5 Sectioning using the SBFSEM System

Sectioning using SBFSEM System

In this section, the ultramicrotome’s cutting performance in terms of minimum slice

thickness and cut surface roughness was verified and demonstrated. Alternative ways to

improve cut surface roughness is discussed. Finally, a sample TEM grid was embedded

in resin, then sectioned and 3D reconstructed to demonstrate the accuracy of the

ultramicrotome.

5.1 Specimen Height Control for Sectioning

To control the height of the specimen for sectioning and avoid having to wait until the

settling time of the actuator each time, a larger step input can be given to the actuator and

the cuts can be taken at the appropriate time. The transient characterization in 4.3 can be

used. For example, if a step input of 500 nm is set, then five 100 nm cuts can be taken

within the settling time (20.5 min), as shown in Figure 5.1.

Figure 5.1: Timings for five cuts in 20.5 min, with a 500 nm step input.

1.17 min, 100 nm

2.67 min, 200 nm

4.8 min, 300 nm

8.42 min, 400 nm

20.5 min, 500 nm

0

100

200

300

400

500

0 5 10 15 20 25

Actu

ato

r positio

n [

nm

]

Time [min]

38

Choosing this step input amount allows ample time for the SEM image capture

between the 100 and 200 nm cuts. Typically, a capture of a high-quality image in SEM

takes approximately 30 seconds.

5.2 Minimum Slice Thickness

The minimum slice thickness that the designed SBFSEM system was tested to ensure

that it met the minimum slice thickness requirement of 100 nm. The ultramicrotome can

cut 90 nm slices 95% of the time.

To test the thickness, a block of EMbed 812 resin prepared with a 350 x 350 µm2 block

face area was prepared to be cut with a 45° glass knife. As the specimen was advanced

little by little according to the characterization data in section 4.3, the knife was moved

over to attempt a cut each time. If a cut was made, the section would fall on the block face

or pile on the knife edge. A failure occurs either if an entire slice was not removed from

the block face, or if no cutting occurred at all. Failures are likely caused by the fact that

the knife edge is not sharp enough, causing it to “roll over” as it tries to cut.

5.3 Cutting Surface Roughness

A low surface roughness after cutting, meaning a smooth surface, means that the

cutting thickness is uniform. During development, interim results showed that increasing

the stiffness of the ultramicrotome structure greatly decreased surface roughness. This

observation guided the ultramicrotome design to become stiff. To ensure the chatter

during cutting is low, the surface roughness is measured. The results are shown in Figure

5.2.

39

EMbed 812 blocks were prepared with a 350 x 350 µm2 block face area. The slicing

thickness was 200 nm. After cutting, the block face is scanned by atomic force microscopy

(AFM) to obtain the roughness profile of the surface. When sectioning, chatter marks will

appear running parallel to the knife edge, while marks caused by nicks in the knife edge

appear running perpendicular to the knife edge. The AFM scans are run perpendicular

to the knife edge to measure the chatter. A sample cut using an older revision of the

ultramicrotome having insufficient stiffness is also included for comparison.

Figure 5.2: (Top left and right) Optical micrographs of a rough (A) and smooth (B) block face. The

cutting direction is from top to bottom. The rough block face was cut using an older revision of the

ultramicrotome which was not as stiff and severe chatter occurred. (Bottom) The AFM line scans of the

sample show that the RMS of the smooth surface is substantially lower than the RMS of the rough

surface.

Rough profile (A):RMS: 122 ± 8.3 nm

Smooth profile (B):RMS: 15.7 ± 2.4 nm

-300

-200

-100

0

100

200

300

400

500

600

0 10 20 30 40 50 60 70 80 90

Devia

tion [

nm

]

x [µm]

40

The AFM scan showed that the surface roughness of the block face cut using the

newest revision of the ultramicrotome is substationally smoother, having an RMS surface

roughness of 15.7 nm compared to 122 nm cut using an older revision of the

ultramicrotome. The stiffness of the new ultramicrotome is approximately 50 times that

of the older version.

5.4 Cutting Parameters Affecting Surface Roughness

The following describes effects of the parameters of knife cutting speed and slicing

thickness on surface roughness were studied to find acceptable ranges, i.e. giving less

than 20 nm roughness RMS. These tests were performed cutting an EMbed 812 sample

with a block face area of 350 x 350 µm2 using a glass knife. Each slice was 200 nm thick.

Results showed that at ranges tested, knife speed had little effect on chatter, while cutting

too thick will cause lots of chatter, as shown in Figure 5.3.

Figure 5.3: (Left) There is little to no effect of knife speed on chatter. Statistical analysis shows a low

correlation value of -0.45 between knife speed and chatter. (Right) As section thickness is increased,

little chatter until a section thickness of 500 nm, when the roughness RMS rapidly rises.

Average RMS: 16.6 nm

0

5

10

15

20

25

30

35

40

0 200 400 600 800

Roughness R

MS

[nm

]

Knife Cutting Speed [µm/s]

0

10

20

30

40

50

60

70

80

0 200 400 600

Ro

ugh

nes

s R

MS

[nm

]

Slice thickness [nm]

Too thick

41

5.4.1 Knife speed has little effect on chatter

Typical knife cutting speeds in other SBFSEM ultramicrotomes are in the range of 0.1

to 1 mm/s [37]. Running the knife too fast or too slow during cutting may cause chatter

during sectioning, as discussed in section 2.3. When the knife is run too fast, besides

causing chatter due to undampened vibrations, the great force of the crash of the knife

edge into the sample may cause knife edge chipping or other damage to the sample itself.

In our tests, a range of speeds from 0.1 to 0.7 mm/s were tested, which are within the

typical cutting speed range [37]. The results in this range showed that there is little to no

effect of knife speed on chatter, which had an average value of 16.6 nm, shown in Figure

5.3.

5.4.2 Chatter increases as slice thickness increases

The slice thickness range of this ultramicrotome with acceptable chatter is from 90 nm

to 400 nm. A sample was prepared similarly to in 5.4.1, and was cut at varying slice

thicknesses with a knife speed of 0.25 mm/s. The results are shown in Figure 5.3.

The lower limit of slice thickness testing was the result shown in section 5.2. As

discussed in section 2.3, when the slice is too thick, the force required to curl the section

over the knife blade is high and causes chatter. This happens because the force will start

to pull the knife down into the block face until it is deep enough that the elastic force of

the ultramicrotome stiffness pulls the knife back to the surface. This process repeats and

its periodicity produces the distinct chatter. Although slices thicker than 500 nm are

sometimes cut in conventional ultramicrotomy, in SBFSEM, the section thicknesses are

usually lower. Higher structure stiffness should increase the thickness that the

ultramicrotome can cut smoothly.

42

5.5 Cutting Force Estimation for Future Design Guideline

Following the results of the effects of slice thickness on chatter presented in 5.4.2, the

cutting force may be estimated to define a threshold of the minimum required stiffness

to withstand a certain cutting force. The estimation is based on the cutting mechanics,

which is briefly discussed in 2.3.

To estimate the cutting force, fracture energies of resin reported in [71] and section

compression values reported by [51] can be used with the cutting geometry presented in

[55] shown in Equation 5.1 and Equation 5.2. An introduction to cutting geometry is given

in 2.3 and a detailed discussion is given in [55]. The implication of this is that knowing

the material properties of a material to be cut and the geometry of the knife, one can

estimate the required stiffness of the structure required to withstand the cutting force.

In Equation 5.1, r is compression ratio, where t1 is the thickness before and t2 is the

thickness after compression. The rake angle is α and the slip plane is φ. In Equation 5.2,

Wc is work expended to section per unit volume, F is the force along the upper knife face,

Fc is the force of the knife parallel to the block surface face, Ft is the force of the knife

Equation 5.1: Rake angle (α) and the compression

ratio (r) are used to calculate the slip plane (φ).

𝑟 =𝑡1

𝑡2=

sin(𝜑)

cos (𝜑 − 𝛼)

Equation 5.2: Work expended to section per unit volume (Wc), rake angle (α), and slip plane

angle (φ) are used to calculate the vertical (Ft) and lateral (Fc) forces involved in sectioning.

(

𝐹𝑐

𝐹𝑐

𝑊𝑐𝐴0

) = (

𝑐𝑠𝑐 (𝛼) −𝑐𝑜𝑡 (𝛼) 0

0 𝑡𝑎𝑛 (𝜑) 𝑠𝑒𝑐(𝜑)𝑟 0 𝑠𝑖𝑛 (𝜑)

) (

𝐹

𝐹𝑡

𝐹𝑠

)

43

pulling the knife into the block face, and A0 is the cross sectional area of the section before

cutting.

Using these formulae, it was estimated that the stiffness to cutting force ratios to

ensure < 20 nm surface roughness are:

Lateral (Fc): 58.5 N/µm stiffness to 75 mN cutting force

Vertical (Ft): 2.21 N/µm stiffness to 77.4 mN cutting force

Therefore, in the future, this result may be used as a guideline for designing SBFSEM

ultramicrotomes. Along with allowing cutting materials requiring higher force, higher

stiffness is expected to decrease surface roughness overall. This result also reinforces the

idea that reducing the required cutting force may decrease block face roughness.

5.6 Oscillating Knife: A Method to Reduce Cutting Force

Besides changing the angles of the knife, it is also possible to reduce the cutting force

by oscillating the knife along the direction of the knife edge [51], as shown in Figure 5.4.

This method was successfully implemented to decrease slice compression in a

conventional ultramicrotome. By reducing the cutting force, it may be easier to design a

structure stiff enough to withstand the cutting forces incurred while sectioning larger

areas, or harder materials to reduce chatter.

Instead of the ploughing motion as described in orthogonal cutting, oscillating the

knife produces a slicing motion and the apparent bevel angle is smaller as the sample

moves over the knife. Assuming the clearance angle is constant, the apparent rake angle

will increase. The higher the ratio of the knife’s lateral speed caused by vibration to the

knife feed speed, the smaller the apparent bevel angle.

44

Preliminary results were obtained by slicing a 400 nm thick section while vibrating

the knife at 7 kHz as shown in Figure 5.4 (bottom left). An older revision of the

ultramicrotome using a piezoelectric (soft) specimen actuator was used during this test.

Figure 5.4: (Top left) A piezoelectric stack is attached to a knife with a counter weight to oscillate the

knife. (Top right) Knife apparent angle, caused by knife oscillation combined with the knife feed, is

smaller than the knife bevel angle. (Bottom left) Block face after slicing off 400 nm at a frequency of 7

kHz. (Bottom right) Vibrating glass knife becomes damaged easily, causing severe chatter. The left side

of the blade is more damaged than the right side.

45

A knife speed of 0.1 mm/s, as used in this case, gave the smoothest result. 7 kHz was

chosen as a frequency because that was the resonant frequency of the combination of the

piezoelectric stack combined with the counterweight and knife, maximizing the

amplitude. Compared to the block face in Figure 5.2A, it is much smoother, but the

chatter marks are still apparent when compared to Figure 5.2B. The chatter is also

apparent compared to the block face in the 400 nm case in Figure 5.3.

In the final design of the ultramicrotome, the vibrating knive is not used. The

ultramicrotome could not slice thinner than 400 nm with the vibrating knife. The reason

for this may be that the vibration is not totally in line with the knife edge. In fact, the glass

knife edge is not totally straight, whereas in [51] a diamond knife is used which is known

to have a straighter knife edge than glass knives. When cutting while vibrating the knife

at other speeds, there appears to be a shredding action, which may be evidence that the

vibration is out of alignment with the edge.

A consequence of the out-of-alignment vibration is that the knife is more easily

damaged as it oscillates while cutting. Although the force appears to be decreased,

showing less chatter, the longevity of the knife is also decreased. As the knife is cutting,

the misaligned vibrations cause chips to form in the knife edge. A result of cutting using

the damaged knife edge is shown in Figure 5.4 (bottom right), having severe chatter due

to increased cutting force. In this case, the knife was more damaged on the left side than

the right.

This particular arrangement of the vibrating knife leaves the counterweight as an

overhanging mass on the piezoelectric stack. Combined with possible misalignments, the

large counterweight mass, and the high frequency vibration, this could introduce large

tensile stresses in the stack, leading to premature stack failure. However, the results show

promise that the vibrating knife could be incorporated into future designs.

46

The most obvious solution to both the vibration misalignment and overhanging mass

problem that could be implemented in future designs is a flexure arrangement which

could constrain the vibration to a single direction, as well as preload the stack as to

prevent tensile loading. Other considerations are to determine the modes of vibration of

the flexure to ensure that any off-axis vibration modes are not excited. Each glass knife is

also slightly different, so a change to a diamond knife may be necessary.

5.7 3D Reconstruction Accuracy Validation

To demonstrate the function of the ultramicrotome, a copper TEM grid (Gilder

G2000HS) was embedded in a resin (EMbed812) then sliced, imaged, and 3D

reconstructed as shown in Figure 5.5. TEM grids have a regular and well- known

structure, allowing us to easily verify the reconstructed image’s integrity. This TEM grid

has nominal dimensions of 12.5 µm grid spacing, with a hole width of 7.5 µm and a

thickness of 6 µm. For 3D reconstruction, 200 slices were cut from a block face of 350 µm

x 300 µm with a knife speed of 0.25 mm/s each at 100 nm thick. The SEM used was the

Hitachi SU3500 using 15 kV accelerating voltage with 30 Pa chamber pressure under

backscatter composition imaging mode.

After images were acquired, they were manually aligned by matching features in the

image that don’t move and cropped to show only the area of interest. Image processing

was performed to remove noise from surrounding areas caused by SEM imaging or

debris falling on the block face. The images were stacked using ImageJ and reconstructed

using the 3DViewer plugin. After cropping, the total volume reconstructed was 87 µm x

67 µm x 20 µm, which is comparable to volumes reconstructed in Table 1.1.

47

In the construction, there are a few pieces of large debris that were left on the block

face, leading to the artifacts outlined in blue in Figure 5.5 (top right). These pieces were

difficult to isolate from the actual TEM grid, so they were not removed. Debris piled on

the knife edge occasionally falls onto the block face. There is also a large discontinuity in

Figure 5.5: (Top left) Individual SEM images were taken, then processed and aligned to form a

stack. (Top right) A TEM grid was reconstructed using SBFSEM with a voxel size of 66 x 66 x

100 nm. The grid spacing as measured from the 3D reconstruction by our SBFSEM system is

matches with manufacturer specifications. Shaded in areas show the first section. Outlined areas

show debris and discontinuities, as labelled. (Bottom) Reference TEM grid with dimensions.

48

the piece shown outlined in orange in Figure 5.5 (top right). The discontinuity occurred

when the cutting process was interrupted because the SEM beam emitter had burned out

and needed replacement. A mistake was made upon restarting the sectioning process. An

interesting note is that although it is not recommended to take more than 50 slices on one

area of a glass knife [9], this particular knife was used for more than 200. This knife may

be an outlier.

Measurements shown in Figure 5.5 (top right) compared with reference dimensions

from Figure 5.5 (bottom) show that the ultramicrotome was able to 3D reconstruct a

sample with good accuracy. The two squares that were measured were picked because

they were far from any debris. The results are accurate within 1% and are presented in

Table 5.1.

Table 5.1: 3D reconstruction is accurate within 1% for length measurements.

Dimension Reference (nm) Measured (nm) Error (%)

Hole width 7540 ± 5 7518 ± 5 -0.29

Hole pitch 12500 ± 5 12627 ± 49 1.0

The measured dimension is outside the error range of the reference. One reason could

be because of the warping of the TEM grid, which is not flat even before it is embedded

in the resin. Measuring on a warped model will introduce small amounts of errors. Also,

the curvature of the knife blade, although small, may have contributed to the error. The

reconstruction was done assuming each slice is perfectly flat. There are also SEM errors.

Over time, the brightness, focus, and position of the SEM beam will drift. Although

efforts were made to align the images in post-processing manually, it is reasonable to

expect approximately 1-2 pixels (66-132 nm) of error when aligning by eye. The errors

observed are within this range.

49

In conclusion, the SBFSEM ultramicrotome is good for morphology visualization, as

shown by the 3D reconstruction, and it was accurate (< 1% error) in length measurements.

However, the large amount of SEM image drift required manual alignment which may

introduce errors in measurements. In the future impelementing computer vision

algorithms like template matching can compensate for this drift and increase accuracy.

50

Chapter 6 Conclusion and Outlook

Conclusion and Outlook

This thesis presented the development of a prototype of a plug-and-play SBFSEM

system that can be used to study morphology in biological and materials science. It was

accomplished by using compact actuators that are typically used for SEM

nanomanipulation.

Plug-and-Play SBFSEM Ultramicrotome

A prototype plug-and-play SBFSEM ultramicrotome was designed that can be used

with no modification to the SEM. By reducing the size of the ultramicrotome using

smaller piezoelectric and thermal actuators, it can be used with any SEM that has the

required imaging modes. It has the capability to perform 3D reconstruction of large

volumes for studies in materials or biological sciences. Because it is plug and play, it can

be installed and uninstalled easily and quickly, meaning it will not disrupt shared

electron microscopy facilities.

Piezoelectric Flexure Actuator

A piezoelectric flexure actuator is designed that has high motion range and a high

out-of-plane stiffness. The high motion range ensures that the knife can slice the entire

surface of the sample. The high stiffness knife actuator will not deflect too much while

cutting, ensuring that there is low chatter.

Thermal V-beam Actuator

A macro-scale version of a MEMS v-beam actuator was designed and characterized

to be used as a specimen advance actuator. Instead being heated by current running

through its flexure beams, the actuator is heated by a cartridge heater installed in its tip.

51

The fact that the thermal expansion of the flexure beams provides the displacement for

the actuator means that it can be much stiffer than a piezoelectric flexure actuator of the

same size.

3D Reconstruction of a TEM grid

The SBFSEM ultramicrotome’s function was verified by sectioning and reconstructing

a TEM grid embedded in resin. The morphology of the grid was preserved in the

reconstruction. Measurements of the reconstructed grid’s dimensions were accurate

within 1% of the grid’s specifications, showing that the ultramicrotome has good

accuracy.

6.1 Contributions

1. Designed and characterized a prototype plug-and-play SBFSEM ultramicrotome.

2. Developed a method to improve out-of-plane stiffness of sagittal piezoelectric

flexure mechanisms.

3. Developed a macro-scale MEMS-style v-beam thermal actuator.

4. Demonstration of function of ultramicrotome, sectioning and 3D reconstructing a

TEM grid with high accuracy.

6.2 Future Directions

Add closed loop control to specimen actuator. This allows the specimen to be raised or

lowered more quickly and accurately.

1. Optimize the arrangement of the actuators to increase the stiffness and reduce the

size, possibly make it load lock compatible so it can be used in high-vacuum SEMs.

52

2. Implement computer vision algorithms to compensate for drift in SEM image or

to align images for more accurate 3D reconstruction.

3. Develop a flexure arrangement allowing a vibrating knife to lower the required

cutting force.

4. Implement an auto-approach feature to the specimen advancing to the knife,

lowering the risk of damaging the knife and to reduce operator mental load.

53

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